Modeling and Functional Characterization of Reconstituted Efflux Pump Components from Heterologous Gram-Negative...

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Antimicrobial resistance (AMR) is a growing global health threat driven by bacterial evolution.
• Tripartite resistance nodulation division (RND) efflux pumps contribute significantly to AMR by extruding antibiotics.
• Components of these efflux pumps can be transferred between bacterial species and genera.

Purpose of the Study:

• To investigate the structural and functional conservation of distantly related efflux pump proteins.
• To demonstrate the functionality of assembled efflux pumps from different Gram-negative bacteria.
• To explore the interaction between RND efflux pumps and quorum-sensing molecules.

Main Methods:

• Assembly of tripartite efflux pump components (MFP and RND) from *E. coli*, *P. aeruginosa*, and *K. pneumoniae*.
• Real-time Nile Red assays to detect efflux pump activity.
• Docking and molecular dynamics (MD) simulations to study protein interactions.

Main Results:

• Evidence of functioning efflux pumps demonstrated through Nile Red assays.
• Enhanced biofilm formation observed in bacteria expressing assembled efflux pumps.
• Molecular simulations revealed direct interactions between RND efflux pumps and AI-2 quorum-sensing molecule.

Conclusions:

• Conserved structural and functional properties of RND efflux pumps facilitate AMR.
• Assembled efflux pumps contribute to antibiotic resistance and biofilm formation.
• Efflux pump components, potentially transferred via horizontal gene transfer, drive AMR and bacterial adaptation.